Abstract 600: Novel Assays to Determine in vivo Platelet Activation
In vivo platelet activation is associated with several pathologic entities, including acute coronary syndrome, essential thrombocythemia, anti-phospholipid antibody syndrome, diabetes and metabolic syndrome. The need for more robust biomarkers with which to assess platelet activation in vivo in human diseases is well recognized. Our lab has developed two novel assays to study in vivo platelet activation and rate of turnover.
During platelet activation, malondialdehyde (MDA) is produced by the thromboxane synthase in amounts equimolar to thromboxane A2 and also non-enzymatically by lipid peroxidation resulting from the oxidative processes that accompany activation. MDA is a reactive dicarbonyl that reacts with amines, notably lysines on proteins, yielding covalent modifications of the proteins that then accumulate over the lifetime of the platelet. We developed an LC/MS/MS method for quantification of the most stable of three MDA adducts, the dilysyl-MDA crosslink, employing a [13C12] labeled internal standard. We found that activation of platelets with arachidonic acid leads to an increase in the levels of dilysyl-MDA crosslinks in platelets, which is inhibited by the thromboxane synthase inhibitor, ozagrel, by the cyclooxygenase inhibitor, aspirin, and by scavengers of reactive carbonyls, 3- methoxysalicylamine and Salicylamine.
High platelet turnover has been associated with increased risk for thrombosis and failure of antiplatelet agents. We propose a novel approach to studying platelet turnover by labeling platelets in vivo by oral administration of aspirin containing a deuterium labeled acetyl group (d3-aspirin). We can measure the clearance of d3-labeled platelets by using LC/MS/MS to measure the tryptic peptide (SLK) of COX-1 labeled with d3. This approach avoids exposure to radioactivity and the artifact resulting from manipulation of platelets labeled ex vivo.
The results of this study provide two novel assays with the potential to serve as markers of in vivo platelet activation and turnover, which may be useful in predicting thrombotic risk and efficacy of antiplatelet therapy in patients with medical conditions associated with platelet hyperactivity and high rate of platelet turnover.
Author Disclosures: I.R. Sosa: Research Grant; Significant; AHA 13FTF17400011. I. Zagol-Ikapitte: None. M. Bala: None. O. Boutaud: None. J. Oates: None.
This research has received full or partial funding support from the American Heart Association.
- © 2015 by American Heart Association, Inc.